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Ploquin, M. et al. Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases. Nucleic Acids Res. 35, 2719-2733

Genome Stability Laboratory, Laval University Cancer Research Center, Hôtel-Dieu de Québec, 9 McMahon, Quebec city, QC, Canada G1R 2J6.
Nucleic Acids Research (Impact Factor: 9.11). 03/2007; 35(8):2719-33. DOI: 10.1093/nar/gkm174
Source: PubMed

ABSTRACT Genetic analysis of fission yeast suggests a role for the spHop2-Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2-Mnd1 binds single-strand DNA ends of 3'-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca(2+) alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.

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    • "Hop2–Mnd1 is most effective in synaptic complex assembly and in the D-loop reaction when present at amounts ∼1/4–1/15 the concentration of DMC1 (26,27,29,31). Cytological analysis has revealed that the majority of Hop2 and Mnd1 proteins do not co-localize with DMC1 in S. cerevisiae and A. thaliana cell nuclei during meiosis (16,17,19,47). "
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    ABSTRACT: The Hop2-Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2-Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2-Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2-Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.
    Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt924 · 9.11 Impact Factor
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    • "Previous biochemical studies revealed that the human, mouse and yeast Hop2–Mnd1 complexes (17–21), the yeast Swi5–Sfr1 complex (22), human RAD54B (14,23,24) and RAD51AP1 (25) stimulate homologous pairing mediated by Dmc1. These proteins also stimulate the Rad51-mediated homologous pairing (17,18,20,22,26–28). Meiotic cells produce both Dmc1 and Rad51; however, the functional difference between them has not been elucidated. "
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    ABSTRACT: PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.
    Nucleic Acids Research 12/2011; 40(7):3031-41. DOI:10.1093/nar/gkr1229 · 9.11 Impact Factor
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    • "Ca 2+ does not support formation and BM of HJs in the four-strand exchange reaction Ca 2+ stimulates D-loop formation and DNA strand exchange by human Rad51 and Dmc1 (Bugreev and Mazin 2004; Bugreev et al. 2005), and also stimulates D-loop formation by fission yeast Dmc1 (Ploquin et al. 2007). "
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    ABSTRACT: Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5-Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex-duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5'-to-3' direction and Rad51 promotes exchange in the 3'-to-5' direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production.
    Genes & development 03/2011; 25(5):516-27. DOI:10.1101/gad.1997511 · 12.64 Impact Factor
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